Development and characterization of segment-specific enteroids from the pig small intestine in Matrigel and transwell inserts: insights into susceptibility to porcine epidemic diarrhea Virus.

Introduction: The critical early stages of infection and innate immune responses to porcine epidemic diarrhea virus (PEDV) at the intestinal epithelium remain underexplored due to the limitations of traditional cell culture and animal models. This study aims to establish a porcine enteroid culture model to investigate potential differences in susceptibility to infection across segments of the porcine small intestine (duodenum, jejunum, and ileum). Methods: Intestinal crypt cells from nursery pigs were cultured in Matrigel to differentiate into porcine enteroid monolayer cultures (PEMCs). Following characterization, PEMCs were enzymatically dissociated and subcultured on transwell inserts (PETCs) for apical surface exposure and infection studies. Characterization of region-specific PEMCs and PETCs included assessment of morphology, proliferation, viability, and cellular phenotyping via immunohistochemistry/immunocytochemistry and gene expression analysis. Subsequently, PETCs were inoculated with 105 TCID50 (50% tissue culture infectious dose)/mL of a high pathogenic PEDV non-S INDEL strain and incubated for 24 h. Infection outcomes were assessed by cytopathic effect, PEDV N protein expression (immunofluorescence assay, IFA), and PEDV N-gene detection (quantitative reverse transcription polymerase chain reaction, RT-qPCR). Results: No significant morphological and phenotypical differences were observed among PEMCs and PETCs across intestinal regions, resembling the porcine intestinal epithelium. Although PETCs established from different segments of the small intestine were susceptible to PEDV infection, jejunum-derived PETCs exhibited higher PEDV replication, confirmed by IFA and RT-qPCR. Discussion: This segment-specific enteroid culture model provides a reliable platform for virological studies, offering a controlled environment that overcomes the limitations of in vivo and traditional cell culture methods. Standardizing culture conditions and characterizing the model are essential for advancing enteroid-based infection models.

Field efficacy of a recombinant toxoid vaccine against Shiga toxin 2e during a naturally occurring edema disease infection.

The objective of this field trial was to determine the efficacy of a recombinant toxoid vaccine against Shiga toxin 2e (Stx2e) in piglets suffering from edema disease (ED). Three farms with confirmed ED cases were selected for the field trials. On each farm, a total of 40 4-day-old pigs were randomly allocated to either the vaccinated or unvaccinated group, with 20 pigs per group (10 males and 10 females). A 1.0-mL dose of the recombinant toxoid vaccine was administered intramuscularly to pigs in the vaccinated groups in accordance with the manufacturer's recommendations at 4 d of age. A single 2.0-mL dose of phosphate-buffered saline was administered to unvaccinated pigs at the same age. Clinical signs of ED were observed in 12 piglets in the unvaccinated groups and 7 unvaccinated pigs died as a result of ED out of the total number of piglets on Farms A, B, and C. Vaccination had a positive effect on pig growth performance compared to that of unvaccinated pigs on 2 of the 3 farms. Seroconversion of neutralizing antibodies against Stx2e occurred in 70% of piglets in the vaccinated group on Farm A, 75% of vaccinated piglets on Farm B, and 55% of vaccinated piglets on Farm C, when detectable antibodies were measured at 17 d post-vaccination (dpv). Detectable antibodies were measured in 85% of vaccinated piglets on Farms A and B and in 90% of these piglets on Farm C at 37 dpv. These field trials determined that the ED recombinant toxoid vaccine successfully reduced clinical signs and mortality, improved average daily weight gain, and resulted in the production of neutralizing antibodies against Stx2e in pigs.

L'objectif de cette étude sur le terrain était de déterminer l'efficacité d'un vaccin à base d'anatoxine recombinante contre la toxine Shiga 2e (Stx2e) chez les porcelets souffrant de la maladie de l'oedème (ED). Trois fermes ayant des cas confirmés de ED ont été sélectionnées pour les études sur le terrain. Dans chaque ferme, un total de 40 porcs âgés de 4 jours ont été répartis au hasard dans le groupe vacciné ou non vacciné, avec 20 porcs par groupe (10 mâles et 10 femelles). Une dose de 1,0 ml du vaccin à base d'anatoxine recombinante a été administrée par voie intramusculaire aux porcs des groupes vaccinés conformément aux recommandations du fabricant à l'âge de 4 jours. Une dose unique de 2,0 ml de solution saline tamponnée au phosphate a été administrée aux porcs non vaccinés au même âge. Des signes cliniques de ED ont été observés chez 12 porcelets des groupes non vaccinés et 7 porcs non vaccinés sont morts des suites d'une maladie de l'oedème sur le nombre total de porcelets des fermes A, B et C. La vaccination a eu un effet positif sur les performances de croissance des porcs par rapport à celles des porcs non vaccinés dans 2 des 3 fermes. La séroconversion des anticorps neutralisants contre Stx2e s'est produite chez 70 % des porcelets du groupe vacciné de la ferme A, 75 % des porcelets vaccinés de la ferme B et 55 % des porcelets vaccinés de la ferme C, lorsque des anticorps détectables ont été mesurés 17 jours après la vaccination (dpv). Des anticorps détectables ont été mesurés chez 85 % des porcelets vaccinés des fermes A et B et chez 90 % de ces porcelets de la ferme C à 37 dpv. Ces études sur le terrain ont déterminé que le vaccin toxoïde recombinant ED réduisait avec succès les signes cliniques et la mortalité, améliorait le gain de poids quotidien moyen et entraînait la production d'anticorps neutralisants contre Stx2e chez les porcs.(Traduit par Docteur Serge Messier).

A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

Integrin αVß3 mediates porcine deltacoronavirus infection and inflammatory response through activation of the FAK-PI3K-AKT-nf-κB signalling pathway.

Porcine deltacoronavirus (PDCoV) is an emerging porcine enteropathogenic coronavirus that causes acute watery diarrhoea in piglets, resulting in significant economic losses to the global swine industry. However, the underlying mechanism of PDCoV infection is not well defined, which seriously hinders the development of effective drugs and vaccines. Integrins (ITG) are heterodimeric transmembrane glycoproteins that play important roles in the life cycle of many viruses. In the current study, the viral entry pathways of PDCoV were explored and the role of ITGαVß3 was investigated during PDCoV infection. Our results showed that the lysosomal acidification inhibitor bafilomycin-A1 (Baf-A1) significantly reduced PDCoV infection, while exogenous protease facilitated PDCoV infection and even allowed PDCoV entry to bypass the endosomal pathway, suggesting PDCoV entry into cells via the endocytic pathway and the exogenous protease-mediated pathway simultaneously. Furthermore, ITGαVß3 was identified to be involved in PDCoV infection, especially during viral entry stages. PDCoV infection triggers the activation of the focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-serine/threonine-specific protein kinase (AKT) signalling pathway, and this activation is ITGαVß3-dependent, suggesting that the activation of the FAK-PI3K-AKT signalling pathway during PDCoV infection is mediated by ITGαVß3. Our results further demonstrated that PDCoV infection induced the expression of inflammatory cytokines, which was mediated by activation of the ITGαVß3-FAK-PI3K-AKT-nuclear transcription factor-κB (NF-κB) signalling pathway. Overall, the results revealed that ITGαVß3 is an essential host factor for PDCoV infection and can serve as a supplementary receptor to facilitate PDCoV infection, which can help us to explore the molecular mechanism of PDCoV infection.

Identifying the host factors required for entry will be helpful in uncovering the pathogenesis mechanisms and developing antivirals against the emerging coronavirus porcine deltacoronavirus (PDCoV). Herein, we revealed that PDCoV enters cells via the endocytic and exogenous protease-mediated pathways simultaneously. Integrins (ITG) αVß3 is a host factor required for PDCoV infection, especially during virus adhesion, invasion, and release. Most importantly, PDCoV promotes viral infection by activating the ITGαVß3-focal adhesion kinase (FAK)-phosphatidylinositol 3-kinase (PI3K)-serine/threonine-specific protein kinase (AKT) signalling pathway and induces inflammation by activating the ITGαVß3-FAK-PI3K-AKT-NF-κB signalling pathway. Overall, this is the first study to identify ITGαVß3 as an essential factor for PDCoV infection, which can help us to confirm the molecular regulatory mechanism and provide a comprehensive resource for PDCoV infection.

Development and Validation of Serotype-Specific Blocking ELISA for the Detection of Anti-FMDV O/A/Asia1/SAT2 Antibodies.

Foot-and-mouth disease (FMD) is one of the most infectious viral transboundary diseases of livestock, which causes devastating global economic losses. Different enzyme-linked immunosorbent assays (ELISAs) are used for sero-surveillance of the foot-and-mouth disease virus (FMDV). However, more sensitive, accurate, and convenient ELISAs are still required to detect antibodies against FMDV serotypes. The primary goal of this study was to establish serotype-specific monoclonal antibody (mAb)-based blocking ELISAs (mAb-bELISAs) that would provide better performance characteristics or be equivalent in performance characteristics compared with a conventional polyclonal antibody (pAb)-based competitive ELISA (pAb-cELISA). Four mAb-bELISAs were developed using FMDV serotype-specific mAbs for the detection of anti-FMDV/O/A/Asia1/SAT2 antibodies. Using a 50% cut-off, all four mAb-bELISAs exhibited species-independent 99.74%, 98.01%, 96.59%, and 98.55% diagnostic specificity (DSp) and 98.93%, 98.25%, 100%, and 87.50% diagnostic sensitivity (DSe) for FMDV serotypes O, A, Asia1, and SAT2, respectively. In addition, a 100% DSe of serotypes O- and SAT2-specific mAb-bELISAs was observed for porcine sera when the cut-off was 30%. All mAb-bELISAs developed in this study displayed high repeatability/reproducibility without cross-reactivity. Finally, the diagnostic performance of mAb-bELISAs was found to be better than or equivalent to compared with pAb-cELISAs, suggesting that mAb-bELISAs can be used to replace existing pAb-ELISAs for the detection of antibodies against these four FMDV serotypes.

Evaluation of circulating immune cells, analytes, and inflammatory markers in sows affected with postpartum dysgalactia syndrome.

Postpartum dysgalactia syndrome (PDS) is a condition affecting periparturient sows, characterized by a reduction in milk and colostrum synthesis shortly after farrowing. Insufficient milk production results in substantial economic losses due to increased piglet morbidity/mortality and premature sow culling. Since PDS develops within a few days following farrowing, the study objectives were to determine if periparturient immune cell profiles and circulating biomarkers differ in sows affected by PDS. We hypothesized differences in immune cells, circulating analytes, and inflammatory markers would exist at farrowing in sows that subsequently developed PDS compared to healthy herd-mates. Thirty-six sows with PDS symptoms were matched by parity and day of lactation with 36 healthy control (CON) sows. Diagnosis of PDS (timepoint 2) occurred on average 9.25 ±â€…2.67 d after farrowing. Blood samples and litter weights were collected at farrowing (timepoint 1) and at the onset of clinical PDS (timepoint 2). Piglets from PDS sows had lower average daily gain and higher mortality than piglets from CON (P < 0.01). Aspartate aminotransferase was increased (20%; P ≤ 0.06) in PDS sows compared to CON at both timepoints. Additionally, blood urea nitrogen was increased in PDS sows at timepoint 1 and timepoint 2 (13%; P = 0.08 and 16%; P = 0.01, respectively). At timepoint 2, total protein, globulin, magnesium, and cholesterol were increased (P ≤ 0.03) while γ-glutamyl transferase and albumin were decreased (P ≤ 0.02) in PDS sows. Lipopolysaccharide-binding protein, an inflammatory biomarker, was increased (48%; P = 0.07) at timepoint 2 in PDS compared to CON sows. Collectively, these data indicate PDS sows have altered metabolism and appear immune activated compared to healthy herd-mates, and further investigation is needed to determine if PDS can be predicted at farrowing.

Postpartum dysgalactia syndrome (PDS) is a multifactorial disorder affecting periparturient sows characterized by a pronounced reduction (dysgalactia) in milk secretion. Insufficient milk production limits piglet growth, leading to increased piglet mortality and often removal of affected sows from the herd, ultimately compromising sow longevity and negatively impacting the profitability of swine operations. The objective of this study was to determine if differences in circulating immune cells, analytes, and inflammatory markers exist at farrowing in sows that subsequently develop PDS compared to healthy herd-mates. Thirty-six sows with PDS were matched by parity and day of lactation with 36 healthy control (CON) sows. Blood samples were collected at farrowing (timepoint 1) and at the onset of clinical PDS (timepoint 2). Differences in markers of tissue catabolism (blood urea nitrogen, ß-hydroxybutyrate, aspartate aminotransferase, alanine aminotransferase) and inflammation (lipopolysaccharide-binding protein, haptoglobin, albumin) were observed in PDS sows compared to control, suggesting PDS sows have altered metabolism and are potentially immune activated compared to healthy herd-mates.

Lawsonia intracellularis regulates nuclear factor-κB signalling pathway during infection.

Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) in pigs, horses and wide range of mammals. Little is known about the role of innate immune response during L. intracellularis infection. In this study, we investigated the nuclear factor-κB (NF-κB)-regulated immune response against infection of a clinical strain Dkp23 and a live-attenuated Enterisol vaccine strain in PK-15 cells. We found that expression of NF-κB target genes TNF-α, IFN-γ, IL-6 and IL-8 were modulated during the course of infection. At 5 dpi, there was a significant increase in p65 NF-κB activation, including protein nuclear translocation and phosphorylation, synchronous with the induction of IL-6, IFN-γ and IL-8 expression in L. intracellularis infected cells, especially for Enterisol vaccine strain-infected cells. This result suggests that NF-κB signalling level is induced when L. intracellularis bacterial load peaks at 5 dpi. The induction of pro-inflammatory cytokines expression is consistent with the decreased viability of L. intracellularis-infected cells especially that of the vaccine strain. There were no significant changes in NF-κB signalling between vaccine and Dkp23 infection in PK-15 cells, except for moderate levels of differences in NF-κB target genes expression which might be a reflection of differences in intracellular bacterial load. Overall, the data presented here indicate a correlation between the induction of NF-κB signalling and the L. intracellularis bacterial load in PK-15 cells.

The genetic resistance of sows to Escherichia coli F4 adhesion reduces their response to a vaccine containing F4 fimbriae but does not affect the preweaning performance of their susceptible piglets.

INTRODUCTION: Pigs without intestinal receptors for F4 fimbriae are congenitally resistant to F4 fimbriae-bearing enterotoxigenic Escherichia coli (ETEC F4). In general, 50 % and 100 % of piglets born to resistant (RR) sows crossed with hetero- or homozygous susceptible (SR, SS) boars, respectively, are susceptible but do not receive colostral antibodies against F4 fimbriae unless the sows have been vaccinated. The question arises as to whether resistant sows produce protective amounts of F4 antifimbrial antibodies after vaccination. The serum and colostrum antibody titres of 12 resistant and 12 susceptible vaccinated gilts were compared. The effect of the receptor status of the dam and sire on the preweaning performance of 5027 piglets was evaluated using Agroscope's recordings. The sows of the experimental herd, where ETEC F4 was circulating, were vaccinated against ETEC twice during the first pregnancy and once during each following pregnancy. The log2 transformed F4 antibody titres in the serum obtained after the second vaccine injection as well as in the colostrum of the 12 resistant animals were lower than the titres of the susceptible animals (serum: F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019; colostrum: F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). The heat labile enterotoxin (LT) antibody titres after vaccination did not differ between susceptible and resistant animals (p > 0,10). Preweaning mortality in the offspring of RR sows × SS boars was slightly lower than in the offspring of SS sows × RR boars (P = 0,04), suggesting that the disease risk of susceptible piglets born to vaccinated resistant sows was not increased, even though they received colostrum with a slightly reduced content of antibody against F4 fimbriae.

INTRODUCTION: Les porcs dépourvus de récepteurs intestinaux pour les fimbriae F4 sont congénitalement résistants aux Escherichia coli entérotoxinogènes porteurs de fimbriae F4 (ETEC F4). En général, 50 % et 100 % des porcelets nés de truies résistantes (RR) croisées avec des verrats hétéro- ou homozygotes sensibles (SR, SS), respectivement, sont sensibles mais ne reçoivent pas d'anticorps colostraux contre les fimbriae F4, à moins que les truies n'aient été vaccinées. La question se pose de savoir si les truies résistantes produisent des quantités protectrices d'anticorps antifimbriae F4 après la vaccination. Les titres d'anticorps dans le sérum et le colostrum de 12 truies reproductrices vaccinées résistantes et de 12 truies reproductrices vaccinées sensibles ont été comparés et l'effet du statut récepteur de la mère et du père sur les performances avant sevrage de 5027 porcelets a été évalué. Les truies du troupeau expérimental, où circulait ETEC F4, ont été vaccinées deux fois au cours de la première gestation et une fois au cours de chaque gestation suivante contre ETEC. Les titres d'anticorps F4 transformés en log2 dans le sérum obtenu après la deuxième injection de vaccin ainsi que dans le colostrum des 12 animaux résistants étaient inférieurs aux titres des animaux sensibles (sérum : F4ab 11,19 ± 1,44 vs. 12,18 ± 1,33, P = 0,096 ; F4ac 10,03 ± 1,58 vs. 11,59 ± 1,43, P = 0,019 ; colostrum : F4ab 12,20 ± 2,41 vs. 14,02 ± 1,31, P = 0,033 ; F4ac 10,93 ± 2,46 vs. 13,03 ± 5,21, P = 0,006). Les titres d'anticorps contre l'entérotoxine thermolabile (LT) après la vaccination ne différaient pas entre les animaux sensibles et résistants (p > 0,10). La mortalité avant sevrage dans la progéniture des truies RR × verrats SS était légèrement inférieure à celle de la progéniture des truies SS × verrats RR (P = 0,04), ce qui suggère que le risque de maladie des porcelets sensibles nés de truies résistantes vaccinées n'a pas été augmenté, même s'ils ont reçu du colostrum avec une teneur légèrement réduite en anticorps contre les fimbriae F4.

The immunoglobulin M-degrading enzyme of Streptococcus suis (IdeSsuis) leads to long-lasting inhibition of the activation of porcine IgM-secreting B cells.

Streptococcus suis (S. suis) is one of the most important porcine pathogens, causing severe pathologies such as meningitis or polyarthritis. It is also a very successful colonizer of mucosal surfaces. The IgM-degrading enzyme of S. suis (IdeSsuis) specifically cleaves porcine IgM, which results in complement evasion. On the basis of our previous finding that IdeSsuis also cleaves the IgM B cell receptor in vitro, we verified IgM B cell receptor cleavage ex vivo in whole regional lymph nodes and investigated the working hypothesis that this IgM B cell receptor cleavage results in a long-lasting impaired B cell function. The number of IgM-secreting cells was determined via ELISpot analysis after porcine peripheral blood mononuclear cells had initially been treated with different recombinant S. suis proteins and subsequently stimulated with interleukin-2 and the toll-like receptor 7/8 ligand R848. Compared with treatment with medium or recombinant muramidase-released protein, treatment with rIdeSsuis but also with a cleavage-deficient variant led to a reduction in the number of IgM-secreting cells as well as the level of secreted IgM. Flow cytometry analysis confirmed that the IgM B cell receptor was cleaved only by rIdeSsuis, and the receptor recovered to pretreatment levels on day 2 after treatment. Flow cytometry analysis of B and T cells incubated with fluorescein-labelled recombinant proteins revealed that different rIdeSsuis variants bind specifically to B cells, most prominently the cleavage-deficient variant. Our results indicate that in vitro interference of rIdeSsuis with the IgM B cell receptor results in long-lasting impaired IgM secretion by B cells after toll-like receptor activation. Further studies are warranted to prove that the modulation of B cell function by IdeSsuis could play a role in vivo.

Identification of Nonsynonymous SNPs in Immune-Related Genes Associated with Pneumonia Severity in Pigs.

We previously showed that several polymorphisms in genes encoding pattern recognition receptors that cause amino acid substitutions alter pathogen recognition ability and disease susceptibility in pigs. In this study, we expanded our analysis to a wide range of immune-related genes and investigated polymorphism distribution and its influence on pneumonia in multiple commercial pig populations. Among the polymorphisms in 42 genes causing 634 amino acid substitutions extracted from the swine genome database, 80 in 24 genes were found to have a minor allele frequency of at least 10% in Japanese breeding stock pigs via targeted resequencing. Of these, 62 single nucleotide polymorphisms (SNPs) in 23 genes were successfully genotyped in 862 pigs belonging to four populations with data on pneumonia severity. Association analysis using a generalized linear mixed model revealed that 12 SNPs in nine genes were associated with pneumonia severity. In particular, SNPs in the cellular receptor for immunoglobulin G FCGR2B and the intracellular nucleic acid sensors IFI16 and LRRFIP1 were found to be associated with mycoplasmal pneumonia of swine or porcine pleuropneumonia in multiple populations and may therefore have wide applications in the improvement of disease resistance in pigs. Functional analyses at the cellular and animal levels are required to clarify the mechanisms underlying the effects of these SNPs on disease susceptibility.

Development of two recombinant vaccines against Clostridioides difficile infection and immunogenicity in pregnant sows and neonatal piglets.

INTRODUCTION: Clostridioides difficile is the main cause of antibiotic-associated diarrhea in humans and is a major enteropathogen in several animal species. In newborn piglets, colonic lesions caused by C. difficile A and B toxins (TcdA and TcdB, respectively) cause diarrhea and significant production losses. OBJECTIVE: The present study aimed to develop two recombinant vaccines from immunogenic C-terminal fragments of TcdA and TcdB and evaluate the immune response in rabbits and in breeding sows. Two vaccines were produced: bivalent (rAB), consisting of recombinant fragments of TcdA and TcdB, and chimeric (rQAB), corresponding to the synthesis of the same fragments in a single protein. Groups of rabbits were inoculated with 10 or 50 µg of proteins adjuvanted with aluminum or 0.85 % sterile saline in a final volume of 1 mL/dose. Anti-TcdA and anti-TcdB IgG antibodies were detected in rabbits and sows immunized with both rAB and rQAB vaccines by ELISA. The vaccinated sows were inoculated intramuscularly with 20 µg/dose using a prime-boost approach. RESULTS: Different antibody titers (p ≤ 0.05) were observed among the vaccinated groups of sows (rAB and rQAB) and control. Additionally, newborn piglets from vaccinated sows were also positive for anti-TcdA and anti-TcdB IgGs, in contrast to control piglets (p ≤ 0.05). Immunization of sows with the rQAB vaccine conferred higher anti-TcdA and anti-TcdB responses in piglets, suggesting the superiority of this compound over rAB. CONCLUSION: The synthesized recombinant proteins were capable of inducing antibody titers against C. difficile toxins A and B in sows, and were passively transferred to piglets through colostrum.

Vaccination with a Lawsonia intracellularis subunit water in oil emulsion vaccine mitigated some disease parameters but failed to affect shedding.

Lawsonia intracellularis is the causative agent of ileitis in swine that manifests as slower weight gain, mild or hemorrhagic diarrhea and/or death in severe cases. As an economically important swine pathogen, development of effective vaccines is important to the swine industry. In developing a subunit vaccine with three recombinant antigens - FliC, GroEL and YopN - we wanted to identify a formulation that would produce robust immune responses that reduce disease parameters associated with Lawsonia intracellularis infection. We formulated these three antigens with four adjuvants: Montanide ISA 660 VG, Montanide Gel 02 PR, Montanide IMS 1313 VG NST, and Montanide ISA 61 VG in an immunogenicity study. Groups vaccinated with formulations including Montanide ISA 660 VG or Montanide ISA 61 VG had significantly more robust immune responses than groups vaccinated with formulations including Montanide Gel 02 PR or Montanide IMS 1313 VG NST. In the challenge study, animals vaccinated with these antigens and Montanide ISA 61 VG had reduced lesion scores, reduced lesion lengths, and increased average daily gain, but no reduction in shedding relative to the control animals. This work shows that this vaccine formulation should be considered for future study in a field and performance trial.

Porcine epidemic diarrhea virus: A review of detection, inhibition of host gene expression and evasion of host innate immune.

As one of the most important swine enteropathogenic coronavirus, porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute and devastating enteric disease that causes lethal watery diarrhea in suckling piglets. Recent progress in studying PEDV has revealed many intriguing findings on its prevalence and genetic evolution, rapid diagnosis, suppression of host gene expression, and suppression of the host innate immune system. Due to the continuous mutation of the PEDV genome, viral evasions from innate immune defenses and mixed infection with other coronaviruses, the spread of the virus is becoming wider and faster, making it even more necessary to prevent the infections caused by wild-type PEDV variants. It has also been reported that PEDV nsp1 is an essential virulence determinant and is critical for inhibiting host gene expression by structural and biochemical analyses. The inhibition of host protein synthesis employed by PEDV nsp1 may contribute to the regulation of host cell proliferation and immune evasion-related biological functions. In this review, we critically evaluate the recent studies on these aspects of PEDV and assess prospects in understanding the function of PEDV proteins in regulating host innate immune response and viral virulence.

The Role of Wild Boar as Host of Japanese Encephalitis Virus in the Absence of Domestic Pigs.

Pigs are the most common amplifying hosts of the Japanese encephalitis virus (JEV). In 2016, four residents on Tsushima Island who did not own pig farms were diagnosed with JE. Therefore, a serosurvey was conducted to estimate the risk and seroprevalence of JEV after the outbreak. Sera collected from 560 Tsushima Island residents between January and September 2017 were tested for neutralizing antibodies against JEV strains JaGAr01 (genotype 3) and Muar (genotype 5). Sera collected from six wild boars between June and July 2022 were tested. The seroprevalence rates of neutralizing antibodies against JaGAr01 and Muar were 38.8% and 24.6%, respectively. High anti-JEV neutralizing antibody titers of ≥320 were identified in 16 residents, including 3 younger than 6 years with prior JEV vaccination, 2 in their 40s, and 11 older than 70. However, no anti-JEV-specific IgM was detected. Residents who engaged in outdoor activities had higher anti-JEV antibody titers. Sera from wild boars were negative for JEV RNA, but four of six samples contained neutralizing antibodies against JEV. Therefore, JEV transmission continues on Tsushima Island, even in the absence of pig farms, and wild boars might serve as the amplifying hosts.

Transcriptome Analysis Reveals Novel Inflammatory Signalings to Glaesserella parasuis Infection.

Glaesserella parasuis (GPS) can cause severe systemic inflammation in pigs, resulting in huge economic losses to the pig industry. At present, no effective method is available for the prevention and control of GPS infection. Molecular breeding for disease resistance is imminent, but disease-resistance genes have not been identified. To study the mechanism of systemic acute inflammation caused by GPS, we established three in vitro infection models (3D4/21 cells, PK15 cells, and PAVEC cells) according to its infection path. There was no significant difference in apoptosis among the three kinds of cells after 12 h of continuous GPS stimulation, while inflammatory factors were significantly upregulated. Subsequent transcriptome analysis revealed 1969, 1207, and 3564 differentially expressed genes (DEGs) in 3D4/21 cells, PK15 cells, and PAVEC cells, respectively, after GPS infection. Many of the DEGs were predicted to be associated with inflammatory responses (C3, CD44, etc.); cell proliferation, growth and apoptosis; gene expression; and protein phosphorylation. Key signaling pathways, including S100 family signaling, bacteria and virus recognition, and pathogen-induced cytokine storm signaling, were enriched based on Ingenuity Pathway Analysis (IPA). Furthermore, a total of three putative transmembrane receptors and two putative G-protein-coupled receptors, namely F3, ICAM1, PLAUR, ACKR3, and GPRC5A, were identified by IPA among the three types of cells. ACKR3 and GPRC5A play pivotal roles in bacterial adhesion, invasion, host immune response and inflammatory response through the S100 family signaling pathway. Our findings provide new insights into the pathological mechanisms underlying systemic inflammation caused by GPS infection in pigs, and they lay a foundation for further research on disease-resistance breeding to GPS.

Concurrent Ascaris infection modulates host immunity resulting in impaired control of Salmonella infection in pigs.

Ascaris is one of the most widespread helminth infections, leading to chronic morbidity in humans and considerable economic losses in pig farming. In addition, pigs are an important reservoir for the zoonotic salmonellosis, where pigs can serve as asymptomatic carriers. Here, we investigated the impact of an ongoing Ascaris infection on the immune response to Salmonella in pigs. We observed higher bacterial burdens in experimentally coinfected pigs compared to pigs infected with Salmonella alone. The impaired control of Salmonella in the coinfected pigs was associated with repressed interferon gamma responses in the small intestine and with the alternative activation of gut macrophages evident in elevated CD206 expression. Ascaris single and coinfection were associated with a rise of CD4-CD8α+FoxP3+ Treg in the lymph nodes draining the small intestine and liver. In addition, macrophages from coinfected pigs showed enhanced susceptibility to Salmonella infection in vitro and the Salmonella-induced monocytosis and tumor necrosis factor alpha production by myeloid cells was repressed in pigs coinfected with Ascaris. Hence, our data indicate that acute Ascaris infection modulates different immune effector functions with important consequences for the control of tissue-invasive coinfecting pathogens.IMPORTANCEIn experimentally infected pigs, we show that an ongoing infection with the parasitic worm Ascaris suum modulates host immunity, and coinfected pigs have higher Salmonella burdens compared to pigs infected with Salmonella alone. Both infections are widespread in pig production and the prevalence of Salmonella is high in endemic regions of human Ascariasis, indicating that this is a clinically meaningful coinfection. We observed the type 2/regulatory immune response to be induced during an Ascaris infection correlates with increased susceptibility of pigs to the concurrent bacterial infection.

A spike-based mRNA vaccine that induces durable and broad protection against porcine deltacoronavirus in piglets.

Coronaviruses (CoVs) are important pathogens for humans and other vertebrates, causing severe respiratory and intestinal infections that have become a threat to public health because of the potential for interspecies transmission between animals and humans. Therefore, the development of safe, effective vaccines remains a top priority for the control of CoV infection. The unique immunological characteristics of vaccines featuring messenger RNA (mRNA) present an advantageous tool for coronavirus vaccine development. Here, we designed two lipid nanoparticle (LNP)-encapsulated mRNA (mRNA-LNP) vaccines: one encoding full-length spike (S) protein and the other encoding the spike ectodomain (Se) from porcine deltacoronavirus (PDCoV). Fourteen days after primary immunization, both mRNA vaccines induced high levels of immunoglobulin G and neutralizing antibodies in mice, with the S vaccine showing better performance than the Se vaccine. Passive immune protection of the S mRNA vaccine in suckling piglets was confirmed by the induction of robust PDCoV-specific humoral and cellular immune responses. The S mRNA vaccine also showed better protective effects than the inactivated vaccine. Our results suggest that the novel PDCoV-S mRNA-LNP vaccine may have the potential to combat PDCoV infection. IMPORTANCE: As an emerging porcine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) has the potential for cross-species transmission, attracting extensive attention. Messenger RNA (mRNA) vaccines are a promising option for combating emerging and re-emerging infectious diseases, as evidenced by the demonstrated efficacy of the COVID-19 mRNA vaccine. Here, we first demonstrated that PDCoV-S mRNA-lipid nanoparticle (LNP) vaccines could induce potent humoral and cellular immune responses in mice. An evaluation of passive immune protection of S mRNA vaccines in suckling piglets confirmed that the protective effect of mRNA vaccine was better than that of inactivated vaccine. This study suggests that the PDCoV-S mRNA-LNP vaccine may serve as a potential and novel vaccine candidate for combating PDCoV infection.

Intranasal delivery of a recombinant adenovirus vaccine encoding the PEDV COE elicits potent mucosal and systemic antibody responses in mice.

Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus that causes substantial economic loss to the global pig industry. The emergence of PEDV variants has increased the need for new vaccines, as commercial vaccines confer inferior protection against currently circulating strains. It is well established that the induction of mucosal immunity is crucial for PEDV vaccines to provide better protection against PEDV infection. In this study, we constructed a recombinant adenovirus expressing the core neutralization epitope (COE) of G2b PEDV based on human adenovirus serotype 5 (Ad5). We evaluated the effects of different administration routes and doses of vaccine immunogenicity in Balb/c mice. Both intramuscular (IM) and intranasal (IN) administration elicited significant humoral responses, including COE-specific IgG in serum and mucosal secretions, along with serum-neutralizing antibodies. Moreover, IN delivery was more potent than IM in stimulating IgA in serum and mucosal samples and in dampening the immune response to the Ad5 vector. The immune response was stronger after high versus low dose IM injection, whereas no significant difference was observed between high and low IN doses. In summary, our findings provide important insights for developing novel PEDV vaccines.IMPORTANCEPorcine epidemic diarrhea (PED) is a highly contagious disease that has severe economic implications for the pork industry. Developing an effective vaccine against PEDV remains a necessity. Here, we generated a recombinant adenovirus vaccine based on Ad5 to express the COE protein of PEDV (rAd5-PEDV-COE) and systematically evaluated the immunogenicity of the adenovirus-vectored vaccine using different administration routes (intramuscular and intranasal) and doses in a mouse model. Our results show that rAd5-PEDV-COE induced potent systemic humoral response regardless of the dose or immunization route. Notably, intranasal delivery was superior to induce peripheral and mucosal IgA antibodies compared with intramuscular injection. Our data provide valuable insights into designing novel PEDV vaccines.

Efficacy comparison in cap VLPs of PCV2 and PCV3 as swine vaccine vehicle.

As one genotype of porcine circovirus (PCV) identified in 2016, PCV3 has brought huge hidden dangers to the global swine industry together with PCV2. Virus-like particles (VLPs) of capsid protein (Cap) of PCV2 serve as an alternative nano-antigen delivery strategy to efficiently induce antiviral immune response against PCV2 and/or other covalently displayed swine pathogens. However, the current understanding is limited on the capability of PCV3 as a nano-vaccine vehicle. Here we systematically compared the characteristics and the immunogenic efficacy of PCV3 Cap (Cap3) and PCV2 Cap (Cap2) in a VLP form. Cap3 VLPs presented higher internalization efficiency into cells and cytokines production compared to those of Cap2. Meanwhile, cross-reactive immunity between Cap3 VLPs and Cap2 VLPs was detected. Furthermore, to evaluate the function of Cap3 VLPs and Cap2 VLPs as vaccine vehicles carrying foreign proteins, the non-structural protein 6 of porcine reproductive and respiratory syndrome virus (PRRSV) was fused to C-terminus of Cap. Cap3-based chimeric particles induced a higher level of nsp6-specific immune response and PRRSV inhibition. Collectively, these self-assembling, Cap-based VLPs offer a compelling platform for enhancing the effectiveness of subunit vaccinations against newly emerging diseases and hold great promise for the development of Cap3-based chimeric subunit vaccines.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.